Silencing GmBIR1 in Soybean Results in Activated Defense Responses

Receptor-like kinases (RLKs) are a large group of pattern recognition receptors (PRRs) and play a critical role in recognizing pathogens, transducing defense signals, and mediating the activation of immune defense responses. Although extensively studied in the model plant Arabidopsis, studies of RLKs in crops, including soybean, are limited. When a BAK1-interacting receptor-like kinase (BIR1) homolog (referred to as GmBIR1 hereafter) was silenced by the BPMV (Bean pod mottle virus)-induced gene silencing (BPMV-VIGS), it resulted in phenotypes that were reminiscent of constitutively activated defense responses, including a significantly stunted stature with observable cell death on the leaves of the silenced plants. In addition, both SA and H2O2 were over-accumulated in the leaves of the GmBIR1-silenced plants. Consistent with this autoimmune phenotype, GmBIR1-silenced plants exhibited significantly enhanced resistance to both Pseudomonas syringae pv. glycinea (Psg) and Soybean mosaic virus (SMV), two different types of pathogens, compared to the vector control plants. Together, our results indicated that GmBIR1 is a negative regulator of immunity in soybean and the function of BIR1 homologs is conserved in different plant species.


Introduction
Pathogen-associated molecular patterns (PAMPs) are conserved microbial components that can be perceived by plasma membrane-localized pattern recognition receptors (PRRs) and trigger immunity [1]. The PRRs constitute the largest gene family (>600 genes) in Arabidopsis [2]. The PRRs include two types of protein families: the receptor-like kinases (RLKs) and receptor-like proteins (RLPs). While a typical RLK contains an extracellular leucine-rich repeat (LRR) domain, a transmembrane domain (TM), and a cytoplasmic kinase domain, an RLP contains only LRR and TM domains but lacks a cytoplasmic kinase domain. The LRR domain is responsible for perception of PAMPs or ligands, and the cytoplasmic kinase domain is involved in intracellular signal transduction [3]. The lack of a kinase domain in a RLP suggests that an RLP needs to pair with RLKs or another cytoplasmic kinase to transduce signals [3].
Some RLKs have been reported to sense PAMPs either from bacteria or fungi and transduce defense signals [4]. The best studied examples are FLAGELLIN SENSING2 (FLS2) and EF-TU RECEPTOR (EFR). FLS2 recognizes the 22 amino acid peptide flg22 from bacterial flagellin, while EFR recognizes an 18 amino acid peptide, elf18, derived from bacterial translation elongation factor EF-TU [5,6]. BAK1 was originally identified as a

Silencing Glyma.18g246400 Results in Stunted Stature and Spontaneous Cell Death in Soybean
Through BPMV-VIGS silencing approach, we successfully identified both positive and negative regulators of defense responses in soybean [20][21][22][23][24][25]. Using the same strategy, we found that when Glyma.18g246400 was silenced, a significantly stunted stature ( Figure 1A) and spontaneous cell death on the systemic leaves were observed for the silenced plants ( Figure 1B). The cell death was further confirmed by Tryplan blue staining, which specifically stains the dead cells. The massive blue patches were observed along the lager veins on the systemic leaves of the silenced plants but not on that of vector control plants (Compare Figure 1C,D). RT-PCR analysis confirmed that the transcript level of Glyma.18g246400 was significantly reduced in the silenced plants ( Figure 1E). Stunted stature and spontaneous cell death are the signature characteristics of the constitutively activated defense responses. Consistent with this, a significantly induced expression of the PR1 gene was observed in the silenced plants ( Figure 1F). These results indicated that Glyma.18g246400 encodes a negative regulator of cell death and defense responses. Blast search with the fulllength cDNA of Glyma.18g246400 revealed that there is a duplicated gene in the soybean genome, Glyma.09g246600, which shares 95% identity with Glyma.18g246400. It has been reported that VIGS can simultaneously silence genes with a homology greater than 85% at the nucleotide level [26,27]. We confirmed that the silenced phenotype observed for the Glyma.18g246400-silenced plants was actually a result of co-silencing both Glyma.18g246400 and Glyma.09g246600 ( Figure 1G). Both Glyma.18g246400 and Glyma.09g246600 share the highest homology with Arabidopsis BIR1 (65%), we thus referred to these two genes as GmBIR1a and GmBIR1b, respectively. Because the bir1 mutant in Arabidopsis displays a constitutive activated defense phenotype (Gao et al., 2009)

Silencing Glyma.18g246400 Results in Stunted Stature and Spontaneous Cell Death in Soybean
Through BPMV-VIGS silencing approach, we successfully identified both positive and negative regulators of defense responses in soybean [20][21][22][23][24][25]. Using the same strategy, we found that when Glyma.18g246400 was silenced, a significantly stunted stature ( Figure 1A) and spontaneous cell death on the systemic leaves were observed for the silenced plants ( Figure 1B). The cell death was further confirmed by Tryplan blue staining, which specifically stains the dead cells. The massive blue patches were observed along the lager veins on the systemic leaves of the silenced plants but not on that of vector control plants (Compare Figure 1C,D). RT-PCR analysis confirmed that the transcript level of Glyma.18g246400 was significantly reduced in the silenced plants ( Figure 1E). Stunted stature and spontaneous cell death are the signature characteristics of the constitutively activated defense responses. Consistent with this, a significantly induced expression of the PR1 gene was observed in the silenced plants ( Figure 1F). These results indicated that Glyma.18g246400 encodes a negative regulator of cell death and defense responses. Blast search with the full-length cDNA of Glyma.18g246400 revealed that there is a duplicated gene in the soybean genome, Glyma.09g246600, which shares 95% identity with Glyma.18g246400. It has been reported that VIGS can simultaneously silence genes with a homology greater than 85% at the nucleotide level [26,27]. We confirmed that the silenced phenotype observed for the Glyma.18g246400-silenced plants was actually a result of co-silencing both Glyma.18g246400 and Glyma.09g246600 ( Figure 1G). Both Glyma.18g246400 and Glyma.09g246600 share the highest homology with Arabidopsis BIR1 (65%), we thus referred to these two genes as GmBIR1a and GmBIR1b, respectively. Because the bir1 mutant in Arabidopsis displays a constitutive activated defense phenotype (Gao et al., 2009), our results indicate that the function of BIR1 homologs is conserved in different plants species.

Both SA and H 2 O 2 Are Over-Accumulated in the GmBIR1-Silenced Plants
SA is a key hormone that positively regulates defense responses against biotrophic pathogens and is over-accumulated in the plants with activated defense responses [20,28]. To examine whether SA over-accumulation occurred in GmBIR1-silenced plants, both free SA and bound SA were quantified, and the levels of free SA and bound SA were 2.46-and 4.1-fold higher in the GmBIR1-silenced plants than in BPMV-0 control plants, respectively (Figure 2A,B). The over-accumulated level of SA suggests that SA is involved in the constitutively activated defense responses observed in GmBIR1-silenced plants.

Both SA and H2O2 Are Over-Accumulated in the GmBIR1-Silenced Plants
SA is a key hormone that positively regulates defense responses against biotrophic pathogens and is over-accumulated in the plants with activated defense responses [20,28]. To examine whether SA over-accumulation occurred in GmBIR1-silenced plants, both free SA and bound SA were quantified, and the levels of free SA and bound SA were 2.46-and 4.1-fold higher in the GmBIR1-silenced plants than in BPMV-0 control plants, respectively (Figure 2A,B). The over-accumulated level of SA suggests that SA is involved in the constitutively activated defense responses observed in GmBIR1-silenced plants. H2O2 is a potent trigger of cell death [29,30] and H2O2 is over-accumulated in the plants with activated defense responses [20,23]. To examine if H2O2 is over-accumulated in the GmBIR1-silenced plants, the leaves collected from both vector control plants and the GmBIR1-silenced plants were stained with 3,3-diaminobenzidine (DAB) [31,32]. More intense brown spots were observed on the leaves of the GmBIR1-silenced plants compared to the BPMV-0 control plants ( Figure 2C), indicating that the cell death observed in the GmBIR1-silenced plants was correlated with increased level of H2O2 accumulation.

Silencing GmBIR1 Enhances Resistance against SMV and Pseudomonas syringae pv. glycinea (Psg), R4 Strain
Activated defense responses are usually correlated with enhanced disease resistance [20][21][22][23][24][25]. To examine whether disease resistance is enhanced in GmBIR1-silenced plants, SMV strain N tagged with the GUS marker protein (SMV-N-GUS) [33] was inoculated via biolistic bombardment onto three individual leaves collected from both the vector control plants and the GmBIR1-silenced plants. Both the size of the GUS foci and the intensity of GUS staining reflect viral infectivity. At 5 days post inoculation (dpi), the H 2 O 2 is a potent trigger of cell death [29,30] and H 2 O 2 is over-accumulated in the plants with activated defense responses [20,23]. To examine if H 2 O 2 is over-accumulated in the GmBIR1-silenced plants, the leaves collected from both vector control plants and the GmBIR1-silenced plants were stained with 3,3-diaminobenzidine (DAB) [31,32]. More intense brown spots were observed on the leaves of the GmBIR1-silenced plants compared to the BPMV-0 control plants ( Figure 2C), indicating that the cell death observed in the GmBIR1-silenced plants was correlated with increased level of H 2 O 2 accumulation.

Silencing GmBIR1 Enhances Resistance against SMV and Pseudomonas syringae pv. glycinea (Psg), R4 Strain
Activated defense responses are usually correlated with enhanced disease resistance [20][21][22][23][24][25]. To examine whether disease resistance is enhanced in GmBIR1-silenced plants, SMV strain N tagged with the GUS marker protein (SMV-N-GUS) [33] was inoculated via biolistic bombardment onto three individual leaves collected from both the vector control plants and the GmBIR1-silenced plants. Both the size of the GUS foci and the intensity of GUS staining reflect viral infectivity. At 5 days post inoculation (dpi), the SMV-N-GUS infection was visualized by GUS staining. The GUS infection foci were easily seen on the vector control leaves ( Figure 3A, left panels; Figure 3B). However, the GUS spots on the GmBIR1-silenced plants were much smaller and only visible under a dissecting microscopy ( Figure 3C). The average diameter of the GUS foci formed on the vector control leaves was four-fold of those formed on the GmBIR1-silenced leaves ( Figure 3D). This result indicates that silencing GmBIR1 leads to increased resistance against SMV.
SMV-N-GUS infection was visualized by GUS staining. The GUS infection foci were easily seen on the vector control leaves ( Figure 3A, left panels; Figure 3B). However, the GUS spots on the GmBIR1-silenced plants were much smaller and only visible under a dissecting microscopy ( Figure 3C). The average diameter of the GUS foci formed on the vector control leaves was four-fold of those formed on the GmBIR1-silenced leaves (Figure 3D). This result indicates that silencing GmBIR1 leads to increased resistance against SMV. We reported that silencing GmFLS2 compromises soybean resistance against Pseudomonas syringae pv. glycinea (Psg) R4 [24]. To test the effect of GmBIR1 silencing on Psg infection, we inoculated the BPMV-0 control plants and the GmBIR1-silenced plants with Psg. At 4-and 6-dpi, the colony forming unit of Psg on the vector control leaves was significantly higher than on that of the GmBIR1-silenced leaves ( Figure 4). Together, these results clearly show that silencing GmBIR1 enhances resistance of soybean plants against two totally different types of pathogens, SMV and Psg.  We reported that silencing GmFLS2 compromises soybean resistance against Pseudomonas syringae pv. glycinea (Psg) R4 [24]. To test the effect of GmBIR1 silencing on Psg infection, we inoculated the BPMV-0 control plants and the GmBIR1-silenced plants with Psg. At 4-and 6-dpi, the colony forming unit of Psg on the vector control leaves was significantly higher than on that of the GmBIR1-silenced leaves ( Figure 4). Together, these results clearly show that silencing GmBIR1 enhances resistance of soybean plants against two totally different types of pathogens, SMV and Psg.
SMV-N-GUS infection was visualized by GUS staining. The GUS infection foci were easily seen on the vector control leaves ( Figure 3A, left panels; Figure 3B). However, the GUS spots on the GmBIR1-silenced plants were much smaller and only visible under a dissecting microscopy ( Figure 3C). The average diameter of the GUS foci formed on the vector control leaves was four-fold of those formed on the GmBIR1-silenced leaves (Figure 3D). This result indicates that silencing GmBIR1 leads to increased resistance against SMV. We reported that silencing GmFLS2 compromises soybean resistance against Pseudomonas syringae pv. glycinea (Psg) R4 [24]. To test the effect of GmBIR1 silencing on Psg infection, we inoculated the BPMV-0 control plants and the GmBIR1-silenced plants with Psg. At 4-and 6-dpi, the colony forming unit of Psg on the vector control leaves was significantly higher than on that of the GmBIR1-silenced leaves ( Figure 4). Together, these results clearly show that silencing GmBIR1 enhances resistance of soybean plants against two totally different types of pathogens, SMV and Psg.

The Activated Defense Responses in GmBIR1-Silenced Plants Are Independent of GmMPK3/6 Activation
Activation of MAPK signaling pathway is one of the signature hallmarks of activated defense responses [34]. To test whether the activated MAPK signaling pathway contributes to the activated defense responses and enhanced disease resistance observed in GmBIR1silenced plants (Figures 1-4), the activation of GmMPK3 and GmMPK6 in response to flg22 was examined both in vector control plants and GmBIR1-silenced plants using the Phospho-p44/42 MAP Erk1/2 antibody that recognizes phosphorylation of Arabidopsis and soybean MPK3/4/6 at the sites corresponding to threonine (T) 202/tyrosine (Y) 204 of human (Homo sapiens) p44 MAPK [23,24,35]. Contrary to our expectation, activation of both GmMPK3 and GmMPK6 by flg22 was significantly reduced in GmBIR1-silenced plants compared to vector control plants (Figure 4), indicating that the activated defense responses observed in the GmBIR1-silenced plants are independent of GmMPK3/6 activation.

GmBIR1 Is Localized at the Plasma Membrane (PM) and in the Nucleus
RLKs normally function at the PM. To investigate the subcellular location of GmBIR1, the full-length GmBIR1a was fused to the C-terminus of GFP and transiently co-expressed in N. benthamiana leaves with a PM marker, AtCBL1-mCherry, via agroinfiltration. As shown in Figures 5 and 6, GFP-GmBIR1 was not only co-localized with AtCBL1-mCherry but also presented in the nucleus, indicating that GmBIR1 has dual subcellular localizations at the PM and in the nucleus.

The Activated Defense Responses in GmBIR1-Silenced Plants Are Independent of GmMPK3/6 Activation
Activation of MAPK signaling pathway is one of the signature hallmarks of activated defense responses [34]. To test whether the activated MAPK signaling pathway contributes to the activated defense responses and enhanced disease resistance observed in GmBIR1-silenced plants (Figures 1-4), the activation of GmMPK3 and GmMPK6 in response to flg22 was examined both in vector control plants and GmBIR1-silenced plants using the Phospho-p44/42 MAP Erk1/2 antibody that recognizes phosphorylation of Arabidopsis and soybean MPK3/4/6 at the sites corresponding to threonine (T) 202/tyrosine (Y) 204 of human (Homo sapiens) p44 MAPK [23,24,35]. Contrary to our expectation, activation of both GmMPK3 and GmMPK6 by flg22 was significantly reduced in GmBIR1-silenced plants compared to vector control plants (Figure 4), indicating that the activated defense responses observed in the GmBIR1-silenced plants are independent of GmMPK3/6 activation.

GmBIR1 Is Localized at the Plasma Membrane (PM) and in the Nucleus
RLKs normally function at the PM. To investigate the subcellular location of GmBIR1, the full-length GmBIR1a was fused to the C-terminus of GFP and transiently co-expressed in N. benthamiana leaves with a PM marker, AtCBL1-mCherry, via agroinfiltration. As shown in Figures 5 and 6, GFP-GmBIR1 was not only co-localized with AtCBL1-mCherry but also presented in the nucleus, indicating that GmBIR1 has dual subcellular localizations at the PM and in the nucleus.

The Function of BIR1 Homologs Is Conserved from Different Plant Species
It has been reported that the extensive cell death, induction of PR genes, enhanced accumulation of SA and H2O2, impaired activation of MPK4, and enhanced resistance to

The Function of BIR1 Homologs Is Conserved from Different Plant Species
It has been reported that the extensive cell death, induction of PR genes, enhanced accumulation of SA and H 2 O 2 , impaired activation of MPK4, and enhanced resistance to different pathogens were observed for Arabidopsis bir1-1 mutant [14,15,[36][37][38]. Similarly, spontaneous cell death, constitutively activated autoimmune responses (including induced PR gene expression), accumulated levels of both SA and H 2 O 2, and enhanced disease resistance to both viral and bacterial pathogens were also observed for the GmBIR1-silenced soybean plants (Figures 1-3). The phenotypic reminiscence between bir1-1 mutant and the GmBIR1-silenced plants strongly indicate that the functions of BIR1 homologs are highly conserved across plant species in negatively regulating cell death and immunity. Given that cell death occurs in bir1-1 mutants even under sterile conditions, BIR1 may not function in perception of PAPMs (pathogen-associated molecular patterns).

The Constitutively Activated Cell Death and Defense Responses in GmBIR1-Silenced Plants Is A Result of Co-Silencing of Both GmBIR1a and GmBIR1b
Soybean is a paleopolyploid and 75% of genes in its genome contain at least two copies [39]. Consistent with this, there are two GmBIR1 genes, GmBIR1a and GmBIR1b, in the soybean genome. The open reading frame sequences of GmBIR1a and GmBIR1b share 95% identity at the nucleotide level. The DNA fragment used for GmBIR1 silencing contains four stretches of sequences with 100% identity between GmBIR1a and GmBIR1b that could potentially generate siRNAs of >20 nt in length (Supplementary Figure S1). Therefore, both GmBIR1a and GmBIR1b were silenced using the same construct ( Figure 1G). The autoimmune phenotype of the GmBIR1-silenced plants ( Figure 1B) actually is a result of cosilencing of both GmBIR1a and GmBIR1b. We have previously shown that multiple copies of GmMPK4, GmMPK6, GmMEKK1, GmFLS2, and GmATG2 genes could be successfully cosilenced using a single BPMV-VIGS construct [20,[22][23][24][25]. Given that soybean is polyploidy and is recalcitrant to transformation, VIGS is currently one of the most efficient approaches to interrogate gene functions in soybean [26,27].

Does the Autoimmunity Observed in GmBIR1-Silenced Plants Result from Activated PTI or Activated ETI?
Activation of either PTI or ETI results in autoimmune responses, although the autoimmune responses resulting from ETI are much stronger and last longer than those resulting from PTI [1]. Structure analysis indicates that Arabidopsis BIR1 and BAK1 directly interact with each other through their ecto-LRR domains [36]. Under no-elicited conditions, BIR1 LRR -BAK1 LRR interaction sequesters BAK1 and prevents formation of a signal competent complex with FLS2. In the presence of flg22, flg22-bound FLS2 LRR outcompetes BIR1 LRR for binding to BAK1 LRR from the BIR1 LRR -BAK1 LRR complex and forms a signaling competent flg22-FLS2 LRR -BAK1 LRR complex [36] Based on this structural study, BIR1 functions to keep BAK1-mediated cell death signaling under tight control, thus preventing undesired autoimmunity, and therefore, it is possible that the autoimmunity in bir1-1 or GmBIR1-silenced plants is a result of activation of BAK1-mediated PTI. Our Psg infection assay supported this possibility (Figure 4). However, inconsistent with this possibility, the flg22-induced activation of GmMPK3 and GmMPK6, which is a hallmark event of activated PTI (Meng et al., 2013), was significantly reduced in GmBIR1-silenced plants relative to vector control plants (Figure 4), and the activation of MPK3 and MPK6 in the Arabidopsis bir1-1 mutant is unchanged compared to WT Col-0 [14]. Therefore, it is still unclear whether the autoimmune responses observed in bir1-1 or GmBIR1-silenced plants are solely due to activated PTI, reflecting the complexity of the involvement of BIR1 in plant immunity.
Several lines of evidence indicate that autoimmune responses in bir1-1 or the GmBIR1silenced plants could result from misactivation of R proteins. Firstly, loss function of BIR1 in Arabidopsis leads to the impairment in the activation of MPK4 by flagellin [14].
The autoimmune phenotype of bir1-1 could be the consequence of the impaired MPK4 activation given that Arabidopsis MEKK1-MKK1/2-MPK4 signaling module negatively regulates the activation of the R proteins SUMM2 and SMN1/RPS6 [40][41][42]. Misactivation of SUMM2 and SMN1/RPS6 result from mutations in MEKK1-MKK1/2-MPK4 pathway leads to autoimmune responses [40,42]. However, GmMPK4 activity cannot be induced either by flg22 or SA, regardless of concentration and duration of treatments [23]. Therefore, it is unclear whether GmMPK4 plays a similar role as its counterpart in Arabidopsis. As the GmMPK4 activation is also not observed in the vector control plants (Figure 4 and [23]), it is unlikely that the GmBIR1-silenced phenotypes are related to GmMPK4 function. Secondly, in Arabidopsis thaliana, the copain-like protein BONZAI1 (BON1) interacts physically with and phosphorylates BIR1 and bir1-1 mutant is partially suppressed by loss of function mutation in the resistance gene SNC1 [18]. Thirdly, the autoimmune phenotype of bir1-1 can be partially suppressed by elevated temperatures [18], which is a characteristic feature of R protein-mediated responses [43,44]. Lastly, the autoimmune phenotype of bir1-1 is rescued by the loss of function of EDS1 and PAD4 [14], two key players in TIR type of R protein activation [45]. These results strongly suggest that the autoimmune phenotype of the bir1-1 mutant is a result of misactivated R protein(s). Therefore, it is possible that BIR1 might participate in both PTI and ETI through interacting with different proteins, and the autoimmune responses observed in bir1-1 and GmBIR1-silenced plants could be combined effects of activated PTI and ETI. The facts that RLKs/RLPs are required for NLR-mediated immunity [46] and RLKs/RLPs and NLRs mutually potentiate each other [47] support the idea that BIR1 may participate in both PTI and ETI.

Is the Enhanced Resistance of GmBIR1-Silenced plants to SMV SA-Dependent?
The enhanced resistance of GmBIR1-silenced plants against Psg could be attributed to the increased accumulation of both SA and H 2 O 2 ( Figure 2) and is thus SA-dependent. The enhanced SMV resistance in GmBIR1-silenced plants may not be solely due to increased accumulation of both SA and H 2 O 2 . In Arabidopsis, Guzman-Benito et al. [38] reported that the enhanced resistance of bir1-1 to Tobacco rattle virus (TRV) is independent of cell death or SA defense priming. The expression of Arabidopsis BIR1 is regulated both at TGS and PTGS level by RNA silencing pathway [38]. A threshold level of BIR1 expression must be properly maintained and an expression beyond the threshold level (either too high or too low) triggers autoimmunity. Whether the enhanced resistance of GmBIR1-silenced plants to SMV is uncoupled from the cell death needs to be further investigated.

Plant Materials
Seeds of soybean (Glycine max 'Williams 82') used in this study were harvested from greenhouse-grown plants previously indexed for the absence of BPMV and SMV [48,49]. Soybean plants were grown in a growth chamber at 22 degrees with a photoperiod of 16 h.

BPMV-Mediated VIGS
BPMV strains, BPMV VIGS constructs, and inoculation of soybean seedlings with DNA-based BPMV constructs via biolistic particle bombardments using a Biolistic PDS-1000/He system (Bio-Rad Laboratories, Hercules, CA, USA) have been described previously [48]. The orthologs of ArabidopsisBIR1 in the soybean genome were identified by BLASTN in phytozome databases. The primers used for construction of the BPMV-GmBIR1 (Glyma.18g246400.1) are: The underlined sequences are BamH I and Kpn I restriction sites, respectively, which were used for directional cloning of the PCR fragment into the BPMV VIGS (IA-D35). The letter C in bold indicates an extra nucleotide in reverse primers needed to maintain the correct open reading frame with BPMV genome.
The GmBIR1a-full primers were used for amplifying the full-length cDNA and the GmBIR1a and GmBIR1b primers are used for determining the specific transcript levels of the GmBIR1a and GmBIR1b, respectively.

Trypan Blue Staining
Cell death was detected specifically by trypan blue staining solution as described by [18]. At 18 dpi with BPMV vector only (BPMV-0) or BPMV-GmBIR1 constructs, second fully expanded soybean trifoliolate leaves counting from the top were detached. The detached leaves were placed in trypan blue staining solution (10 mL glycerol, 10 mL lactic acid, 10 mL water, 10 mg trypan blue powder and 10 g phenol) and boiled for 1-1.5 min. The stained leaves were cleared using tap water and then dipped in the chloral hydrate solution in the shaker overnight to remove the non-specific stains. Images were captured using a stereo microscope (Olympus SZH10, Center Valley, PA, USA).

SA Quantification
SA was quantified using an Agilent 1260 HPLC system (Agilent Technologies) with a diode array detector and a fluorescence detector and a column as described previously [51]. The column was a 4.6-by-75-mm Zorbax SB-C18 with a mobile phase comprising sodium acetate (0.2 m, pH 5.5) and methanol with a series of concentration gradients. The initial methanol gradient was maintained at 3% (v/v) for 12 min, linearly increased to 7% (v/v) at 12.5 min and maintained until 38 min at a flow rate of 0.8 mL min−1 throughout the process. After 1 min, the initial reaction conditions were restored and the system needed to be equilibrated for 7 min before the next injection. Total SA from the sample treated with β-Glucosidase and free SA were detected with a 296 nm excitation wavelength and 410 nm emission wavelength. The concentration was calculated by determining the HPLC peak area according to a standard curve.

H 2 O 2 Detection by DAB Staining
H 2 O 2 was detected by an endogenous peroxidase-dependent in situ histochemical staining procedure using DAB (Sigma-Aldrich, St. Louis, MO, USA) [32]. Detached leaves were placed in a solution containing 1 mg mL−1 DAB (pH 5.5) for 2 h. The leaves were cleared by boiling in 96% (v/v) ethanol for 10 min. H 2 O 2 production was visualized as a reddish-brown precipitate on the cleared leaves [32].

Inoculation of Pseudomonas Syringae pv. Glycinea (Psg) onto the Leaves of Soybean Plants
Psg growth assay was performed as described by [24]. Psg was cultivated at 28 degrees until OD = 1.3. The bacterial culture was centrifuged at 3000 rpm for 10 min and the pellet was re-suspended in double-distilled water with an OD = 1. The vector control or GmBIR1-silenced plants were thoroughly and evenly sprayed with the re-suspended bacterial solution on both upsides and downsides of the leaves. The sprayed plants were immediately covered with transparent plastic bags for at least 24 h.

SMV-N-GUS Inoculation, GUS Staining, and GUS Foci Measurements
At 18 dpi with BPMV vector only (BPMV-0) or BPMV-GmBIR1 constructs, second fully expanded soybean trifoliolate leaves counting from the top were detached and biolistically inoculated with SMV-N-GUS [33,48]. Following SMV-N-GUS inoculation, the detached leaves were put into petri dishes with moist filter papers and kept on a lighted growth shelf for 5 days before GUS staining. GUS staining was performed as described by [52]. Photographs of the leaves with GUS foci were taken using a stereo microscope (Olympus SZH10, Center Valley, PA, USA). The numbers of GUS foci were counted, and the diameters of GUS foci were measured using Soft Image System analysis (IA Package; Olympus).

Western Blot Analysis for Detecting Phosphorylated MPKs
Protein was extracted from soybean leaf tissues using extraction buffer (50 mM Tris-MES pH 8.0, 0.5 M sucrose, 1 mM MgCl 2 , 10 mM EDTA, 5 mM DTT) with protease inhibitor cocktail S8830 (Sigma-Aldrich, St. Louise, MO, USA) added as described by [23]. The extract was centrifuged at 12,000 rpm at 4 • C for 30 min and the supernatant was collected. For immunoblotting, the extracted proteins were separated by SDS-PAGE (10% acrylamide gel) and transferred to PVDF membrane (Millipore, Billerica, MA, USA) by semi-dry electro-transfer (Bio-Rad, Hercules, CA, USA). The membrane was blocked in 1× TBST buffer containing 5% milk powder for 2 h. After washing, the membrane was further incubated with anti-Phospho-p44/p42 MAPK (anti-pTEpY) diluted at 1:3000 (Cell Signaling Technology, Danvers, MA, USA), followed by incubation with secondary antibody diluted at 1:7500. The bands were visualized by incubating with chemiluminescent HRP substrate (Millipore, Billerica, MA, USA).

Subcellular Localization of GmBIR1
The GmBIR1 (Glyma.18g246400.1) open reading frame was amplified by RT-PCR from total RNA extracted from cv Williams 82 soybean plants using the following primers: GmBIR1-PGDG-F-HinDIII: 5 -AAGCTTATGAAGATGTTTATGGGTGGC-3 GmBIR1-PGDG-R-BamHI: 5 -GGATCCTCAATCATGTCCCTCTCGAG-3 The letters in bold represent the cleavage sites for different restriction enzymes. The RT-PCR products were firstly double digested with HindIII + BamHI and subsequently cloned into the PGDG vector [53] to generate the GFP-GmBIR1 fusion construct. These fusion constructs and the AtCBL1-mCherry construct (membrane marker) were coinfiltrated into N. benthamiana leaves as described by [50]. Images were captured with a confocal laser-scanning microscope (Leica TCS SP5 AOBS, Wetzlar, Germany).